Toxicology 292 (2012) 63–70 Contents lists available at SciVerse ScienceDirect Toxicology jou rn al hom epage: www.elsevier.com/locate/toxicol Gene expression and epigenetic changes by furan in rat liver Tao Chen a , Tim D. Williams a , Angela Mally b , Carolin Hamberger b , Leda Mirbahai a , Kevin Hickling c , J. Kevin Chipman a,∗ a School of Biosciences, The University of Birmingham, Birmingham B15 2TT, UK b Department of Toxicology, University of Würzburg, Würzburg D-97078, Germany c AstraZeneca R&D Charnwood Safety Assessment, Loughborough, UK a r t i c l e i n f o Article history: Received 29 September 2011 Received in revised form 25 October 2011 Accepted 27 October 2011 Available online 3 November 2011 Keywords: Furan Rat Liver mRNA DNA methylation miRNA a b s t r a c t Furan, a widely used industrial compound, has been found in a number of heated food items. Furan is car- cinogenic to rats and mice, but the mechanism behind its carcinogenic effect is still not well understood. In this study, we tested the hypothesis that alteration of gene expression relating to cell cycle, apoptosis, DNA damage and of epigenetic modifications including miRNA and DNA methylation may contribute to rodent carcinogenicity of furan. Using quantitative PCR arrays specific to cell cycle-, apoptosis- and DNA damage-related genes, we found that three months furan treatment at 30 mg/kg (5 daily doses per week) induced extensive mRNA expression changes (largely up-regulation) in male Sprague Dawley rat liver, and the gene expression changes did not fully recover after a one month withdrawal of furan. We also found 18 miRNAs were up-regulated and 12 were down-regulated by PCR arrays. Many of these deregulated miRNAs were also found to have similar changes in furan-induced tumour samples. Both hyper- and hypo-methylation of specific gene promoter regions were identified and validated in the 3-month samples and tumour samples by microarray and COBRA (combined bisulfite restriction anal- ysis). No global DNA methylation change was found in the 3 month treatment groups by LC–MS/MS, while furan-induced tumour samples showed global hypomethylation compared to non-tumour tissues. In conclusion, three months furan treatment at a carcinogenic dose resulted in irreversible gene expres- sion changes, miRNA modulation and DNA methylation alteration in combination with a DNA-damage response, which suggests that non-genotoxic mechanisms are important for furan carcinogenicity. © 2011 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Furan is a heterocyclic compound widely used in some seg- ments of the chemical manufacturing industry. It also occurs during the combustion of coal and is a component of tobacco smoke. Furan has been found in a number of heat processed food items such as canned and jarred foods (FDA, 2004). Furan is carcino- genic to rats and mice and was classified as ‘possibly carcinogenic to humans’ (International Agency for Research on Cancer, 1995). Furan-induced rat cholangiocarcinoma (CC) is the most commonly used rodent model. It has been found that >86% incidence of CC was induced in both male and female rats by furan at a dose level of 2 mg/kg b.w. for two years (National Toxicology Program, 1993). Male rats treated with furan at a dose level of 30 mg/kg b.w. for 3 months developed cholangiofibrosis (CF), which progressed to yield 100% incidence of CC in the following 9 or 15 months without further treatment (Maronpot et al., 1991). ∗ Corresponding author. Tel.: +44 1214145422; fax: +44 121 414 5925. E-mail address: j.k.chipman@bham.ac.uk (J.K. Chipman). Little is known about the molecular mechanisms of the develop- ment of furan-induced rat CC. Genetic changes including mutations of K-ras and p53 have been reported in human CC (Arora et al., 1999; Furubo et al., 1999; Itoi et al., 1999; Kang et al., 1999; Tannapfel et al., 2000). However, no K-ras or p53 mutation was found in the furan rodent model (Hickling et al., 2010; Sirica, 1996). Moreover, the existence of a genotoxic mechanism of furan carcinogenicity is controversial. Two recent studies reported that furan was not genotoxic in either in vivo or in vitro studies, and that the major metabolite (cis-2-butene-1,4 dial) was only genotoxic at a concen- tration close to or associated with cytotoxicity (Durling et al., 2007; Kellert et al., 2008). It was therefore suggested that non-genotoxic mechanisms need to be taken into account. During tumour development, the balance between proliferation and apoptosis tends to be disturbed. Indeed, furan-induced cell pro- liferation and apoptosis have been found in mouse and rat liver (Fransson-Steen et al., 1997; Mugford et al., 1997; Wilson et al., 1992) and relevant gene expression changes occur at doses as low as 0.1 mg/kg b.w. (Chen et al., 2010). Furan exposure also leads to irreversible chronic inflammation associated with secondary oxidative DNA damage (Hickling et al., 2010). Thus, alterations in 0300-483X/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2011.10.020